ABSTRACT Inactivation occurs spontaneously after opening in all studied K+ channels, including model channels and the hERG channel that determines timing of the human heart. Inactivation controls channel signaling by determining mean open times and the delay before they can be re-opened, yet its molecular basis remains controversial, with several models proposed. We propose clarify a key aspect distinguishing these models: is K+ ion release from the selectivity filter an essential step in inactivation? Also, it is hypothesized to occur spontaneously because of transmembrane allosteric coupling: intracellular H+-triggered changes in the inner transmembrane helix, TM2, produce the conductive Activated state, but this creates clashes that destabilize the extracellular K+ loaded selectivity filter, and cause it to slowly decay to the K+ depleted state. KcsA, a proton- activated channel, provides a unique opportunity to understand this inactivation process in detail. In contrast to the X-ray diffraction or solution NMR studies, the proposed Solid State NMR studies will be performed on full-length KcsA in hydrated membrane bilayers, using wild type or mutants and varied buffer conditions; hence functional species identified in electrophysiology can be conveniently prepared for NMR. Key signatures for inactivation (mutation dependences, kinetics, and [K+] dependence) confirm that the low pH NMR-detected species is the Inactivated state. Mutants altered in inactivation will be used to further test whether K+ release is essential to inactivation. For several no inactivation is observed, and the dominant species at pH 3-5 is the Activated state. For others inactivation occurs quantitatively, and the dominant species at pH 3-5 is the Inactivated state. Comparing various mutants, correlation between inactivation (by electrophysiology) and K+ depletion (by NMR) will provide a clear test of our hypothesis. An initial study of wild type and E71A provides strong support for this hypothesis. Interconversion rates of the Resting, Activated and Inactivated from electrophysiology will be compared with the K+ release rates from NMR. Our 4D NMR data allow full spectral assignments, and show for the first time that the allosteric coupling operates in both directions: protonation of the pH sensor causes K+ ion release at high ambient [K+], and K+ ion extraction at low [K+] causes pH sensor protonation and opening of TM2 at neutral pH, a novel mechanism for opening a K+ channel. NMR titrations allow quantitative description of the allosteric coupling, clarifying the role of the bilayer, and key amino acids, using coupling-impaired mutants, where bulky residues between the selectivity filter and hinge of TM2 are removed. The structure of the inactivated state has been elusive. NMR spectra of the inactivated state provide an excellent opportunity for determination in bilayers.